The steroid/thyroid hormone nuclear receptor superfamily consists of a large number of transcription factors that regulate a wide variety of cellular processes (reviewed in Mangelsdorf, D. J., et al., Cell 83:835-839, 1995). The most highly conserved region of these proteins is their DNA binding domain (DBD) which contains two zinc finger modules. The DBD of hormone receptors interacts with a six nucleotide core recognition motif, or half-site, resembling the sequence 5′-AGGTCA-3′. Most members of the superfamily homo- and/or heterodimerize with other members of this superfamily, thus binding to two half-sites. The orientation and spacing between the half-sites provides the primary basis for specific DNA binding (reviewed in Glass, C. K., Endocrine Rev. 15:391-407, 1994). However, some members of this superfamily can bind to a single extended half-site version of this sequence (Scearce, L. M., et al., J. Biol. Chem. 268:8855-8861, 1993; Wilson, T. E., et al., Mol. Cell. Biol. 13:5794-5804, 1993; Harding, H. P., and Lazar, M., Mol. Cell. Biol. 15:4791-4802, 1995; Giguère, V., et al., Mol. Cell. Biol. 15:2517-2526, 1995). For example, an optimal binding site for steroidogenic factor 1 (SF-1) contains the sequence 5′-TCAAGGTCA-3′ (Wilson, T. E., et al., supra, 1993).
Ligands for many members of the superfamily have been well-studied; they include the steroid hormones, thyroid hormones, retinoids and vitamin D3. Other members of the superfamily have no known ligand; they are referred to as “orphan” receptors. Orphan receptors can bind DNA as heterodimers, homodimers, or monomers (reviewed in Mangelsdorf, D. J., and Evans, R. M., Cell 83:841-850, 1995).
The human estrogen-related receptor α (hERRα or human ERRα, officially termed NR3B1, Nuclear Receptors Nomenclature Committee, Cell 97:161-163, 1999; in the specification these terms are used interchangeably) is an orphan member of the steroid/thyroid hormone nuclear recepotor superfamily. cDNAs encoding portions of this protein and human ERRβ (officially termed NR3B2 (Cell 97:161-163, 1999); in the specification ERRβ and NR3B2 are used interchangeably) were initially isolated by a reduced-stringency screening of cDNA libraries with probes corresponding to the DBD of the human estrogen receptor α (ERα (officially termed NR3A1 (Cell 97:161-163, 1999)) formerly ER; in the specification, these two terms are used interchangeably) (Giguère, V., et al., Nature 331:91-94, 1988). Using chimeric receptors in transfected cells, Lydon, et al. (Lydon, J. P., et al., Gene Express. 2:273-283, 1992) demonstrated that ERRα contains a transcriptional activation domain. On the basis of amino acid sequence similarity with the receptor SF-1 in part of the DBD, Wilson, et al. (Wilson, T. E., et al., supra, 1993) predicted that ERRα might bind to the extended half-site sequence 5′ TCAAGGTCA-3′ recognized by SF-1. Yang, t al. (Yang, N., et al., J. Biol. Chem. 271:5795-5804, 1996) isolated new cDNA clones encoding most of mouse ERRα by screening a cDNA expression library for proteins capable of binding to sequences present in the promoter region of the lactoferrin gene.
Previously, Wiley, et al. reported the purification of proteins from a HeLa cell nuclear extract that bind the transcriptional initiation site of the major late promoter (MLP) of simian virus 40 (SV40) (Wiley, S. R., et al., Genes Dev. 7:2206-2219, 1993). These proteins, collectively referred to as IBP-s for initiator binding proteins of SV40, were shown to consist of multiple members of the steroid/thyroid hormone receptor superfamily (Wiley, S. R., supra, 1993; Zuo, F., and Mertz, J. E., Proc. Natl. Acad. Sci. USA 92:8586-8590, 1995). Partial peptide sequence analysis indicated that a major component of IBP-s was ERRα (Wiley, S. R., supra, 1993). Thus, at least one binding site for ERRα has been identified in the SV40 late promoter.
To identify functional activities of IBP-s, we performed a variety of genetical and biochemical experiments (Wiley, S. R., supra, 1993; Zuo, F., and Mertz, J. E., supra, 1995). The data from these experiments indicated the following: (i) The SV40 MLP contains at least two high-affinity binding sites for IBP-s situated immediately surrounding (+1 site) and approximately 55 bp downstream of (+55 site) the transcriptional start site. (ii) These high-affinity binding sites include the consensus half-site sequence 5′-AGGTCA-3′. (iii) The binding of IBP-s to these sites results in repression of transcription from the SV40 late promoter both in transfected CV-1 cells (derived from SV40's natural host) and in a cell-free transcription system. (iv) Transfection of CV-1 cells with a plasmid encoding an ERα-ERRα chimeric protein containing all but the first 38 amino acid residues of ERRα results in sequence-specific super-repression of the SV40 late promoter. Thus, ERRα likely possesses the functional activities of IBP-s.
Breast cancer afflicts one in eight women in the United States over their lifetime (Edwards, B. K., et al., Cancer 94:2766-2792, 2002). ERα mediates estrogen responsiveness (reviewed in Sanchez, R., et al., Bioessays 24: 244-254, 2002) and plays crucial roles in the etiology of breast cancer (reviewed in Russo, J., et al., J Natl Cancer Inst Monogr. 27:17-37, 2000). It has been developed into the single most important genetic biomarker and target for breast cancer therapy. ERα is present at detectable levels by ligand-binding and immunohistochemical assays in approximately 75% of clinical breast cancers. Selection of patients with ERα-positive breast tumors increases endocrine-based therapy response rates from about one-third in unselected patients to about one-half in patients with ERα-positive tumors (Clark, G. M. and McGuire, W. L., Breast Cancer Res Treat. 3:S69-72, 1983). Since expression of progesterone receptor (PgR, officially termed NR3C3 (Cell 97:161-163, 1999)) is dependent upon ERα activity, further selection of patients with ERα- and PgR-positive tumors enhances the breast cancer hormonal therapy response rate to nearly 80% (Clark, G. M. and McGuire, W. L., Breast Cancer Res Treat. 3: S69-72, 1983). Although ERβ (officially termed NR3A2 (Cell 97:161-163, 1999)) also mediates responses to estrogens (reviewed in Sanchez, R., et al., Bioessays 24: 244-254, 2002), its roles in breast cancer are not as well understood. Reports have linked ERβ expression with low tumor aggressiveness (Jarvinen, T. A., et al. Am J Pathol. 56:29-35, 2000) and higher levels of proliferation markers in the absence of ERα (Jensen, E. V., et al., Proc Natl Acad Sci. USA 98:25197-15202, 2001).
Members of the ErbB family of transmembrane tyrosine kinase receptors have been implicated in the pathogenesis of breast cancer. The members include epidermal growth factor receptor (EGFR, also HER1; ErbB1), ErbB2 (HER2; Neu), ErbB3 (HER3) and ErbB4 (HER4) (reviewed in Stern, D. F., Breast Cancer Res. 2:176-183, 2000). ErbB members stimulate signal transduction pathways that involve mitogen-activated protein kinase (MAPK). In response to initial binding of epidermal growth factor (EGF)-like peptide hormones, ErbB members form homodimers and heterodimers in various combinations to recruit distinct effector proteins (reviewed in Olayioye, M. A., Breast Cancer Res. 3:385-389, 2001). Although ErbB2 has not been demonstrated to interact directly with peptide hormones, it serves as a common regulatory heterodimer subunit with other ligand-bound ErbB members (reviewed in Klapper, L. N., et al., Proc Natl Acad Sci. USA 96: 4995-5000, 1999; Klapper, L. N., et al., Adv Cancer Res. 77:25-79, 2000). Unlike the other ErbB members, ErbB3 lacks intrinsic kinase activity and, therefore, is required to heterodimerize with other ErbB members to participate in signaling (Guy, P. M., et al., Proc Natl Acad Sci. USA 91: 8132-8136, 1994).
Independent overexpression of either EGFR (reviewed in Klijn, J. G., et al., Endocr Rev. 13:3-17, 1992) or ErbB2 (reviewed in Hynes, N. E. and Stern, D. F. Biochim Biophys Acta. 1198:165-184, 1994) associates with ER-negative tumor status, indicates aggressive tumor behavior, and predicts poor prognosis. Moreover, patients whose tumors coexpress both EGFR and ErbB2 exhibit a worse outcome than patients with tumors that overexpress only one of these genes (Torregrosa, D., et al. Clin Chim Acta. 262:99-119, 1997,Suo, Z., et al. J Pathol. 196:17-25, 2002). Overexpression of ErbB2, most often due to gene amplification, occurs in approximately 15-30% of all breast cancers (Slamon, D. J., et al., Science 235:177-182, 1987), reviewed in (Hynes, N. E. and Stern, D. F., Biochim Biophys Acta. 1198:165-184, 1994). Some (Wright, C., et al., Br J Cancer 65:118-121, 1992; Borg, A., et al., Cancer Lett. 81:137-144, 1994; Newby, J. C., et al., Clin Cancer Res. 3:1643-1651, 1997; Houston, et al., Br J Cancer 79:1220-1226, 1999; Dowsett, M., et al., Cancer Res. 61:8452-8458, 2001; Lipton, A., et al., J Clin Oncol. 20:1467-1472, 2002), but not all reports (Elledge, R. M., et al., Clin Cancer Res. 4:7-12, 1998; Berry, D., et al., J Clin Oncol. 18: 3471-3479., 2000.), have implicated ErbB2 in the development of resistance to antiestrogens.
ErbB2 has been targeted for development of the successful clinical agent Herceptin (trastuzumab), a recombinant humanized monoclonal antibody directed against this receptor's ectodomain (reviewed in Sliwkowski, M. X., et al., Semin Oncol, 26: 60-70, 1999). Herceptin has been shown to be a suitable option as a first-line single-agent therapy (Vogel, C., et al., J Clin Oncol. 20:719-726, 2002), but will likely prove most beneficial as an adjuvant (Slamon, D. J., et al., N Engl J Med. 344:783-792, 2001; Esteva, F. J., et al., J Clin Oncol. 20:1800-1808, 2002). Clinical trials are currently underway to evaluate the combination of Herceptin with antiestrogens as a rational approach to treating ERα-positive/ErbB2-overexpressing tumors (Lipton, A., et al., J Clin Oncol. 20:1467-1472, 2002). In the near future, Herceptin will also likely be evaluated in combination with the small molecule EGFR tyrosine kinase inhibitor ZD1829 (Iressa), since this ATP-mimetic has been shown to almost completely block transphosphorylation of ErbB2 via heterodimerization with EGFR in intact cells (Moulder, S. L., Yakes, et al., Cancer Res. 61:8887-8895, 2001) and inhibits the growth of breast cancer cell lines overexpressing both EGFR and ErbB2 (Normanno, N., et al., Ann Oncol. 13:65-72, 2002). Hence, a combination of ZD1829 and Herceptin may be particularly beneficial to those patients whose tumors coexpress EGFR and ErbB2.
The ability of ErbB3 and ErbB4 to predict clinical course is not as clearly recognized as that of EGFR and ErbB2. ErbB3 has been observed at higher levels in breast tumors than normal tissues, showing associations with unfavorable prognostic indicators including ErbB2 expression (Gasparini, G., et al., Eur J Cancer 1:16-22, 1994), lymph node-positive status (Lemoine, N. R., et al., Br J Cancer 66:1116-1121, 1992), and tumor size. However, it also associated with ERα-positive status, a favorable marker of hormonal sensitivity (Knowlden, J. M., et al., Oncogene 17:1949-1957, 1998). In stark contrast to ErbB2, higher levels of ErbB4 have been associated with ERα-positive status (Knowlden, J. M., et al., Oncogene 17:1949-1957, 1998; Bacus, S. S., et al., Am J Pathol. 148:549-558, 1996), more differentiated histotypes (Kew, T. Y., et al., Br J Cancer 82:1163-1170, 2000) and a more favorable outcome (Suo, Z., et al., J Pathol. 196:17-25, 2002).
Despite the utility of ERs and ErbB members as indicators of clinical course, there remains a great need to identify additional breast cancer biomarkers.